Smarter prepx apollo ngs library prep system

Total RNA from 4 biological replicates of each condition was poly(A)-enriched using Dynabeads Oligo(dT)₂₅ (Thermo Fisher) and fragmented to a mean size of 200-300 nucleotides by incubation in 50 mM MgCl₂ for 8 min at 95 °C. A portion of fragmented RNA was saved as input. Remaining RNA samples were incubated overnight at 4 °C rotating with protein G magnetic beads (Thermo Fisher) coated with EpiMark anti-m⁶A antibody (New England BioLabs). Washes and elution were performed on a Biomek liquid handler (Beckman Coulter). To remove unbound RNA, samples were washed five times with each of the following buffers: reaction buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), low-salt reaction buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), and high salt reaction buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O). RNA was eluted with RLT buffer (Qiagen) and purified with MyOne Silane Dynabeads (Thermo Fisher). RNA libraries for the RNA input, collected supernatant, and IP were constructed using SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer’s protocol. The size distributions of the resulting libraries were assessed using the Tapestation D1000 screen tape (Agilent Technologies), normalized, and sequenced on a NextSeq 550 (Illumina) using a single read 75 cycle kit.

HEK293T cells were transfected with TRM editors combined with non-targeting guide RNAs and total RNA was harvested after 48 h. Total RNA was polyA enriched as described above. Sequencing libraries were prepared using polyA-enriched RNA on a SMARTer PrepX Apollo NGS library prep system (Takara) following manufacturer’s protocols. Libraries were normalized with a Qubit dsDNA HS Assay (Thermo Fisher) and sequenced on a NextSeq 550 (Illumina) using high output v2 kits (Illumina) with 75 cycles. Reads were aligned to a custom hg38 transcriptome containing dCas13 with reference UCSC hg38. Fastq files were filtered with TrimGalore version 0.6.2 (https://github.com/FelixKrueger/TrimGalore) to remove low-quality bases, unpaired sequences, and adaptor sequences. Trimmed reads were aligned to a Homo sapiens genome assembly GRCh38 with a custom Cas13 gene entry by aligning reads with STAR version 2.7.0d (1st pass) followed by a second STAR alignment using generated splice sites from the 1st pass to create a 2-pass STAR Alignment. RSEM version 1.3.1 was used to quantify transcript abundance. The limma-voom package^{66 ,67} was used to normalize gene expression levels and perform differential expression analysis with batch effect correction.